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Acta Pharmacologica Sinica (2011) 32: 1031–1037 npg © 2011 CPS and SIMM All rights reserved 1671-4083/11 $32.00 www.nature.com/aps

Original Article

Combination of , aspirin and as novel paradigm of hormone replacement therapy in rabbit model of

Fa-lin YANG1, #, Ke-qing HU2, #, Xin WANG3, #, Zi-mo LIU4, Qin HU4, Ji-fu LI4, Hong HE4, *

1Clinical Laboratory, Qilu Hospital, Shandong University, Ji-nan 250012, China; 2Department of Cardiology, Ji-nan Central Hospital Affiliated to Shandong University, Ji-nan 250012, China; 3Department of Internal Medicine, the Second Affiliated Hospital, Shan- dong University, Ji-nan 250012, China; 4Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry Of Health, and Department of Cardiology, Qilu Hospital, Shandong University, Ji-nan 250012, China

Aim: To assess a novel hormone replacement therapy (HRT) paradigm using raloxifene, aspirin combined with estrogen in rabbit model of menopause. Methods: Female white rabbits were ovariectomized or sham-operated. The ovariectomized rabbits were divided into 7 groups: valerate (E2), raloxifene, aspirin, E2 /raloxifene, E2/aspirin, E2 /raloxifene/aspirin and vehicle. Two weeks after the operation, the rabbits were administered the above drugs for 12 weeks. Then, the mammary glands were examined histologically, was weighted, and blood sample was collected for analyzing the levels of estrogen, and monocyte chemoattractant protein (MCP)-1, and platelet aggregation. The aortic tissue was examined morphometrically. -1 -1 -1 -1 Results: Compared with E2 0.1 mg·kg ·d treatment alone, the pairing of raloxifene 10 mg·kg ·d with E2 significantly decreased the extent of branches and ducts (5.53%±1.23% vs 15.4%±2.17%, P<0.01), as well as the uterine weight (2.16±0.35 g vs 4.91±0.75 g, P<0.01). However, E2/raloxifene or E2 alone treatment significantly stimulated platelet aggregation relative to vehicle -1 -1 group. Addition of aspirin 5 mg·kg ·d reduced platelet aggregation to almost the same level as the vehicle group. E2 treatment exerted a positive effect on serum lipids and MCP-1, and a regression in aortic intimal plaque size compared to the vehicle. Raloxifene rein- forced the positive effects of E2.

Conclusion: The combination of raloxifene, aspirin and E2 exhibits positive , MCP-1 and atherosclerotic responses with minimal stimulation of and uterine tissues as well as platelet aggregation in a rabbit model of the menopause.

Keywords: estrogen; raloxifene; aspirin; atherosclerosis; hormone replacement therapy

Acta Pharmacologica Sinica (2011) 32: 1031–1037; doi: 10.1038/aps.2011.87; published online 18 Jul 2011

Introduction old assigned to estrogen therapy than in those assigned to pla- The beneficial effects of estrogen are well described and cebo[5]. However, estrogen therapy has also been reported to include reductions in low density lipoprotein be linked with an increased risk of tissue-specific side effects, (LDL-C) and inflammatory cytokines, such as monocyte including uterine , which may result chemoattractant protein-1 (MCP-1), an increase in high den- in uterine , and proliferative effects on mammary tissue, sity lipoprotein cholesterol (HDL-C), and enhanced vascular which may result in an increased risk of [6, 7]. The function, amongst others[1–3]. In animal models, estrogen has ideal hormone replacement therapy (HRT) should reproduce been reported to reduce atherosclerosis[4] and a clinical study the beneficial effects of estrogen without producing these also demonstrated a lower calcified plaque burden in the coro- adverse responses. This concept has led to the development nary arteries of postmenopausal women aged 50 to 59 years of HRT with combined estrogen and progestin, with progestin serving to reverse the endometrial hyperplasia induced by estrogen. However, findings from randomized -con- # These three authors contribute to the paper equally. * To whom correspondence should be addressed. trolled trials in postmenopausal women do not support any E-mail [email protected] cardiovascular benefits of estrogen plus progestin, and dem- Received 2011-01-01 Accepted 2011-05-19 onstrate an increase in the risk of breast cancer[8, 9]. Although npg www.nature.com/aps Yang FL et al 1032

the role of progestin remains poorly defined, preclinical and 2.25±0.20 kg) female New Zealand White rabbits (Agricul- clinical studies have suggested that the co-administration of tural Sciences Institute Products; Ji-nan, China) were used progestin may oppose the cardioprotective effects of estro- in the present study. The rabbits were housed individually gen[10–12]. Furthermore, progestin has no anti-estrogenic effect in standard rabbit cages with a room temperature of 20±2 ºC on mammary tissue and, as a result, women on combined and 12-h light cycle. After 2 weeks of acclimation, bilateral estrogen-progestin treatment had an increase in the relative ovariectomies were performed in 70 rabbits under general risk for breast cancer[8, 9]. anesthesia (sodium pentobarbital 30 mg/kg, intravenously). Continued efforts to provide efficacious HRT with improved All rabbits subsequently received a 1.5% cholesterol diet for safety and for postmenopausal women have gen- 12 weeks. The rabbits were randomized into seven groups erated interest in the development of selective estrogen recep- of 10 rabbits: (A) a placebo-control group receiving vehicle tor (ER) modulators (SERMs). Estrogen typically exhibits an control (saline: 2% Tween 80, 0.5% methylcellulose; OVX+Veh

ER effect in all tissues, whereas SERMs demonstrate group); (B) (E2, Delpharm Lille SAS, Lys- -1 -1 mixed functional activity (ER agonist/antagonist) depending lez-Lannoy, France) 0.1 mg·kg ·d (OVX+E2 group); (C) ral- on the target tissue[13]. To date, no SERM alone has been able oxifene hydrochloride (Lilly SA, Madrid, Spain) 10 mg·kg-1·d-1 to achieve an ideal balance of ER agonist and antagonist activ- (OVX+Ral group); (D) aspirin (Huanghai Company, Shan- ity for an optimal postmenopausal therapy. However, it may dong, China) 5 mg·kg-1·d-1 (OVX+ASA group); (E) raloxifene

be possible to achieve optimal results based on the blended paired with E2 (OVX+E2+Ral group); (F) aspirin paired with

tissue-selective activities of a SERM and estrogen in a novel E2 (OVX+E2+ASA group); and (G) raloxifene and aspirin in

approach. combination with E2 (OVX+E2+Ral+ASA group). Compounds The SERM chosen in this study to test our hypothesis was were administered orally in a saline vehicle to the rabbits. The -1 -1 raloxifene, as it has been confirmed to have estrogen agonist dose of E2 (0.1 mg·kg ·d ) selected was the dose commonly effects on lipids and estrogen antagonist effects on the breast used for research, which has been shown not to cause seri- and uterus[14, 15]. It is also the only SERM being specifically ous disorders in the major organs of cholesterol-fed rabbits[25]. studied for its effects on coronary heart disease events in a The dose of raloxifene (10 mg·kg-1·d-1) was chosen based on prospective randomized controlled trial, which demonstrated previous pharmacokinetic data that showed this dosage gen- that the of coronary events was significantly lower erates a plasma raloxifene concentration comparable with that in postmenopausal women <60 years of age assigned to observed in clinical settings and which reduces aortic accu- raloxifene compared with those assigned to the placebo[16]. mulation of cholesterol in rabbits[26]. Furthermore, it has been Although raloxifene has been reported to be associated with reported that, at a dose of 10 mg·kg-1·d-1, raloxifene is able to [17, 18] -1 -1 an increased incidence of vasomotor symptoms , a previ- counteract the effects of 0.1 mg·kg ·d of E2 and maintain the ous study confirmed that combined use of 17β-estradiol with uterine weight at levels that are indistinguishable from those raloxifene was able to decrease the frequency of vasomotor of vehicle-treated controls in animal tests[27]. Aspirin was symptoms in comparison with raloxifene treatment alone[19]. used at an antithrombotic dosage (5 mg·kg-1·d-1). One group Based on these observations, the pairing of raloxifene with of 10 rabbits was sham-operated, and also received a 1.5% estrogen may represent an attractive therapeutic option. cholesterol diet but no hormone treatment (sham group). The However, estrogen has been reported to increase the risk study protocol was approved by the Animal Care Committee of venous and arterial thrombotic events, such of Shandong University, China and was performed according as [20–22]. Aspirin, one of the most widely used blood- to the Guidelines for the Use of Experimental Animals by the thinning agents in the world, is used long-term in low doses Ministry of Health, China. to prevent heart attacks, and blood clot formation in At the end of study, the rabbits were sacrificed after 12 h people at high risk for these events. In animal tests, low-dose of fasting. Breast tissue specimens were fixed in 10% neutral aspirin has been shown to have a preventive effect on throm- buffered formalin, routinely processed, paraffin embedded, bus formulation[23] and in a , it has been shown to sectioned and stained with hematoxylin and eosin. The per- prevent deep and pulmonary in centage of the extent of gland branches and ducts in the mam- patients postoperatively[24]. mary gland was determined using a computer-based quantita- Therefore, based on the available evidence, it may be postu- tive color image analysis system Ipp6.0 (Media Cybernetics, lated that combined use of raloxifene and aspirin with estro- Bethesda, MD, USA). The acquisition of images and analysis gen might serve as a new treatment paradigm for HRT. To of gland branches and ducts were performed in a blinded determine whether this regimen would result in a physiologi- fashion. Uteri were excised and weighed after removal of cal profile that was distinct from that of estrogen alone, we associated fat and luminal fluids. evaluated breast, uterine, platelet aggregation, lipid, MCP-1 Platelet-rich plasma (PRP) was separated from acid citrate and atherosclerotic lesion responses in a rabbit model of the dextrose-anticoagulated blood. Purity of PRP was validated menopause. by Coulter counter (Qilu Hospital Hematology Laboratory, Shandong, China), yielding <0.1% of leukocyte or red blood Materials and methods cell contamination. The concentration of PRP was adjusted Eighty healthy and sexually mature (age, 3 months; weight, to 5×105/µL. Platelet aggregation to adenosine diphosphate

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(ADP, 10 µmol/L) was performed in PRP by a turbidimetric formaldehyde for 24 h, then rinsed with , stained with method using a whole-blood aggregometer in optical mode Red O for 4 h and rinsed with 70% . The (Model No 560-CA, Chrono-log, Havertown, PA, USA). percentage of aorta staining positively with Oil Red O was

Serum collected from the rabbits underwent E2 measure- determined. Quantification was performed by capturing ment using -linked immunoassay (ELISA). The ELISA images of the aortas with a digital camera and analyzed using kits were purchased from DRG International Inc (Mountain- the Ipp6.0computer-based quantitative color image analysis side, NJ, USA) and the experiments were performed follow- system. A standard size of the aortic segment was used for ing manufacturer’s protocol. The laboratory personnel that all rabbits studied. The acquisition of images and analysis of performed this assay were blinded with respect to any infor- lesions were always performed in a blinded fashion. mation concerning the study groups. All samples were run in Data were expressed as mean±SEM. Inter-group treatment duplicate and the mean values for each sample were used in comparisons were performed with one-way ANOVA with the the analysis. For quality control purposes, positive controls Dunnett test. All analyses were performed using SPSS 13.0 containing known amounts of E2 were included in each batch. software (SPSS, Inc, Chicago, IL, USA). A P-value of 0.05 or This also ensured that the assay values did not dramatically less was considered statistically significant (two-sided). shift over time. The sensitivity was 1.4 pg/mL, the intra-assay co-efficient of variation and inter-assay coefficient of variation Results for all assays were <5%. In the OVX rabbit model, an estrogenic stimulatory response

The biochemical evaluation of serum lipids was carried out was found with E2 treatment in breast tissue. In the OVX+E2 in the same laboratory as the other tests in this study. The group, the alveoli and ducts were dilated to a variable extent, laboratory adhered to the criteria of the World Health Organi- and the percentage of the extent of gland branches and ducts zation Lipid Reference Laboratories. All biochemical exami- in the mammary glands increased significantly compared to nations (serum total cholesterol, HDL-C and ) the OVX+Veh group (15.4%±2.17% vs 4.88%±1.15%, P<0.01). were carried out using a chromatographic enzymatic method No estrogenic responses were found in the OVX+Ral group, in a Technicon automatic analyzer HITACHI-7180 (Hitachi when raloxifene was co-administered with E2 at a dose of 10 -1 -1 High-Technologies , Tokyo, Japan). Serum for the mg·kg ·d (OVX+E2+Ral group), breast stimulation induced measurement of these lipids was harvested immediately after by E2 was reduced to vehicle control levels (5.53%±1.23% vs admission onto the study. LDL-C was calculated using the 4.88%±1.15%, P>0.05). No effect on breast tissue was found Friedwald formula: LDL-C=total cholesterol–HDL-cholesterol with aspirin compared to the OVX+Veh group (Figure 1). [28] –(1/5) triglycerides . The estimation of all lipids was rigor- In the OVX rabbit model, 12-week treatment with E2 at -1 -1 ously quality-controlled by a consultant biochemist (KL), and 0.1 mg·kg ·d (OVX+E2 group) demonstrated a significant frequently checked with the values of another reference labo- increase in uterine weight, which is a surrogate measure for an ratory. Inter-assay and intra-assay variabilities of estimations estrogenic stimulatory response (P<0.01 vs OVX+Veh group). were kept at less than 5%. Associated morphological changes were also observed (data

For the measurement of serum MCP-1, a quantitative sand- not shown). Specifically, E2 resulted in an increase in luminal wich enzyme immunoassay from R&D systems (Abingdon, epithelial hypertrophy compared with vehicle control. No UK) was used, where a monoclonal antibody specific for significant increase in uterine weight was found in OVX+Ral MCP-1 had been pre-coated onto a microplate. Briefly, assay group compared to the OVX+Veh group (2.12±0.4 g vs diluent RD1A plus standard or sample was added to each 2.07±0.34 g, P>0.05), and when raloxifene was co-administered well and left to incubate for 2 h at room temperature. The with E2 (OVX+E2+Ral group), the increase in uterine weight plates were washed four times to eliminate any unbound sub- induced by E2 was reduced to the levels of the vehicle control stances. Then, a conjugate (polyclonal antibody conjugated to group (2.16±0.35 g vs 2.07±0.34 g, P>0.05) (Figure 2). horseradish peroxidase) was added to each well for detection In the OVX rabbit model, 12-week treatment with E2 of the cytokine. After a 2-h incubation at room temperature, (OVX+E2 group) demonstrated a significant increase in the plates were washed four times and substrate solution was whole-blood platelet aggregation compared to the OVX+Veh added to each well. A 20 min incubation at room temperature group (83.8%±6.94% vs 63.1%±8.45%, P<0.05). Aspirin (5 allowed color development in proportion to the amount of mg·kg-1·d-1) decreased the whole-blood platelet aggregation cytokine bound in the initial step. Finally, a stop solution was significantly compared to the vehicle control (P<0.01). When added to each well and the intensity of the coloring measured. aspirin was co-administered with E2 in OVX+E2+ASA group,

The sensitivity was 5.0 pg/mL, the intra- and interassay vari- the stimulation in platelet aggregation induced by E2 was ability was 4.8% and 5.8%, respectively. reduced to almost the same levels as in the OVX+Veh group The aorta was flushed with 250 mL of cold (4 ºC) perfu- (64.5%±7.25% vs 63.1%±8.45%, P>0.05) (Figure 3). sion fixation (4% paraformaldehyde in phosphate buffered The serum estradiol level was much higher in the saline) at 100 mmHg, then removed from the aortic valve to estrogen-treated groups including OVX+E2, OVX+E2+Ral, the iliac bifurcation and carefully cleaned of adhering fat and OVX+E2+ASA, and OVX+E2+Ral+ASA, than in the OVX+Veh connective tissue. The aorta was freed from the adventitia, group (P<0.01). Dyslipidemia was improved in the estrogen opened longitudinally, pinned flat and fixed in 4% para- and/or raloxifene-treated animals (OVX+E2, OVX+E2+Ral,

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Figure 2. Uterine response of E2/raloxifene/aspirin in mature OVX rabbits.

After 12 weeks of treatment, uterine wet weights (g) in response to E2

alone, raloxifene alone, aspirin alone, raloxifene paired with E2, aspirin

and E2, aspirin paired with E2 and combined raloxifene were determined. Compared with sham-operated rabbits, ovariectomy induced significant decreases in uterine wet weights. In OVX rabbits, a significant increase

in uterine weight was observed in the OVX+E2 group, no estrogenic stimulatory effect was found in the OVX+Ral group, when paired with

E2, raloxifene antagonized the E2-induced increase in uterine wet weight to a level similar as that of vehicle control. Mean±SEM. n=10 animals per treatment group. cP<0.01 relative to sham group; fP<0.01 relative i to vehicle control; P<0.01 relative to OVX+E2 group. Veh, vehicle; E2, estradiol valerate; Ral, raloxifene; ASA, aspirin.

Figure 1. Breast response of E2/raloxifene/aspirin in mature OVX

rabbits. After 12 weeks of treatment, breast tissue responses to E2 alone,

raloxifene alone, aspirin alone, E2 plus raloxifene, aspirin and E2, aspirin

paired with E2 and combined raloxifene were analyzed (n=10 animals per treatment group). (A) In OVX rabbits, alveoli and ducts embedded in a loose fibrous stroma with varying amount of fat were variably dilated

in the OVX+E2 group, a regression of the parenchymal lobuloalveolar structures and a replacement of the loose fibrous tissue by fat and dense

fibrous tissue were found in the OVX+E2+Ral group. HE stain, Bar=200 μm. (B) The percentage of the extent of gland branches and ducts in the mammary glands. Mean±SEM. n=10. cP<0.01 relative to sham group; f i Figure 3. Platelet aggregation response of E2/raloxifene/aspirin in P<0.01 relative to vehicle control; P<0.01 relative to OVX+E2 group. Veh, vehicle; E , estradiol valerate; Ral, raloxifene; ASA, aspirin. mature OVX rabbits. After 12 weeks of treatment, whole-blood platelet 2 -1 -1 aggregation (%) in response to E2 (0.1 mg·kg ·d ), raloxifene (10 -1 -1 -1 -1 mg·kg ·d ), aspirin (5 mg·kg ·d ), raloxifene paired with E2, aspirin and

E2, aspirin paired with E2 and combined raloxifene were determined. A OVX+E2+ASA, OVX+E2+Ral+ASA, and OVX+Ral groups) significant increase in platelet aggregation was observed in the OVX+E compared to the vehicle-treated animals (OVX+Veh group). 2 and OVX+E +Ral groups, but not in the OVX+Ral group. Aspirin decreased When raloxifene was co-administered with E , an approximate 2 2 the platelet aggregation significantly as compared to the vehicle control. 15% greater reduction in total cholesterol and a 20% greater When co-administered with E2, the E2-induced increase in platelet reduction in LDL-C relative to E2 alone were observed. There aggregation was reduced to a level with no difference to vehicle control. were no significant differences in body before and Mean±SEM. n=10 animals per treatment group. bP<0.05, cP<0.01 e after treatments (Table 1). relative to vehicle control; P<0.05 relative to OVX+E2 group. Veh, vehicle;

It has been reported that ovariectomy could induce an E2, estradiol valerate; Ral, raloxifene; ASA, aspirin.

Table 1. Changes in serum estradiol, lipids, and weight gain. Mean±SEM. n=10. cP<0.01 vs sham group. eP<0.05, fP<0.01 vs OVX+Veh group. h P<0.05 vs OVX+E2 group.

OVX+E2+Ral Sham OVX+Veh OVX+E OVX+Ral OVX+ASA OVX+E +Ral OVX+E +ASA 2 2 2 +ASA

c f f f f E2 (pg/mL) 76.4±10.8 14.8±2.34 225.4±51.9 18.6±6.35 14.3±4.10 245.7±41.2 223.1±45.2 229.1±39.4 TC (mmol/L) 22.0±4.21 30.1±3.30c 23.5±3.90f 24.7±7.31f 31.3±4.46 19.3±2.13fh 22.6±3.75f 18.2±3.58fh TG (mmol/L) 0.86±0.40 0.92±0.32 0.85±0.44 0.89±0.40 0.90±0.34 0.84±0.48 0.87±0.34 0.85±0.47 LDL-C (mmol/L) 17.1±5.30 26.4±5.03c 16.7±6.23f 20.1±6.61e 25.4±7.81 13.4±3.76fh 15.9±5.46f 12.7±2.75fh HDL-C (mmol/L) 3.67±0.65 2.38±1.0c 3.24±0.90e 3.02±1.06e 2.22±0.41 3.54±0.70e 3.16±0.85e 3.57±0.71e Body weight gain (kg) 0.94±0.12 0.78±0.09 0.82±0.10 0.84±0.08 0.76±0.07 0.87±0.11 0.75±0.07 0.80±0.06

TC, total cholesterol; TG, ; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol.

Acta Pharmacologica Sinica www.chinaphar.com npg Yang FL et al 1035 increase in MCP-1 level[29], and in this study, a significant increase in MCP-1 level was observed after ovariectomy. In OVX rabbits, although reduction in MCP-1 level was not as profound as those associated with E2 (approximately a 40% reduction vs control, P<0.01), a significant decrease in MCP-1 level was observed with raloxifene (approximately a 20% reduction vs control, P<0.05) compared with the OVX+Veh group. When raloxifene was used in combination with E2 in Figure 4. Effects of E2/raloxifene/aspirin on MCP-1 level in mature OVX the OVX+E2+Ral group, a 20% greater reduction in MCP-1 rabbits. After 12 weeks of treatment, serum MCP-1 levels (mmol/L) in level relative to E2 alone was observed (Figure 4). response to E2 alone, raloxifene alone, aspirin alone, raloxifene paired with Histopathological analysis demonstrated that the size of E2, aspirin paired with E2 and combined raloxifene, aspirin and E2 were atherosclerotic lesions in the aorta increased significantly after determined. A significant increase in MCP-1 level was observed after ovariectomy. In the OVX rabbit model, 12-week treatment ovariectomy in the OVX+Veh group compared with the sham group, and -1 -1 -1 -1 both E2 (0.1 mg·kg ·d ) and raloxifene (10 mg·kg ·d ) decreased the MCP- with E2 decreased the size of atherosclerotic lesions signifi- 1 level significantly compared to the vehicle control. When raloxifene was cantly (approximately a 40% reduction vs control, P<0.01). paired with E2, MCP-1 level was reduced by approximately 20% relative Similar results were observed with raloxifene (approximately c to E2 alone. Mean±SEM. n=10 animals per treatment group. P<0.01 a 20% reduction vs control, P<0.05). A slight reduction was relative to sham group; eP<0.05, fP<0.01 relative to vehicle control; observed with aspirin although the difference was not statis- h P<0.05 relative to OVX+E2 group. Veh, vehicle; E2, estradiol valerate; Ral, tically significant compared with vehicle control (P=0.052). raloxifene; ASA, aspirin.

When raloxifene and aspirin were co-administered with E2, an approximate 20% greater reduction relative to E2 alone was seen (Figure 5). profile that was distinct from estrogen alone, and in particular, an improved pharmacological profile compared with estrogen Discussion alone. The purpose of this series of in vivo studies was to establish the The key physiological responses that require close monitor- proof-of-concept for a novel paradigm of HRT. This concept ing in the evaluation of estrogen use are breast and uterine was based on the hypothesis that the combination of ralox- tissue stimulation, as estrogen functions directly through ifene and aspirin with estrogen would result in a physiological the ERs, which are abundant in those tissues. In previous

Figure 5. Effects of E2/ raloxifene/aspirin on atherosclerotic lesion in mature OVX rabbits. After 12 weeks of treatment, atherosclerotic lesions in the aorta (%) in response to E2, raloxifene, aspirin, raloxifene paired with E2, aspirin paired with E2 and combined raloxifene, aspirin and E2 were determined by Oil Red O staining (n=10 animals per treatment group). (A) Oil Red O staining on photographs of aorta. Oil Red O positive area corresponds to lipid deposition. (B) Quantitative analysis of the percentage of Oil Red O positive area (% vessel wall staining positively±SEM). A significant increase in the size of atherosclerotic lesions (Oil Red O staining) was observed after ovariectomy compared with sham-operated animals. In OVX rabbits, although the reduction was not as profound as that associated with E2, a significant decrease was observed in the OVX+Ral group P( <0.05) compared with OVX+Veh group. A mild reduction was observed with aspirin, although the difference was not significant. Further reductions relative to E2 alone were observed c e with E2/raloxifene (approximately 10% vs E2 alone) and E2/raloxifene/aspirin (approximately 20% vs E2 alone). P<0.01 relative to sham group; P<0.05, f h P<0.01 relative to vehicle control; P<0.05 relative to OVX+E2 group. Veh, vehicle; E2, estradiol valerate; Ral, raloxifene; ASA, aspirin.

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studies, ER-antagonistic activity in the breast and uterus has ing assay with E2 alone, raloxifene alone, E2/raloxifene, E2/ [14, 15, 27] been found after raloxifene treatment . In the present aspirin, and E2/raloxifene/aspirin compared with vehicle con- study, serum estradiol levels increased significantly in all the trols in the OVX rabbits. The maximal reduction was observed

estrogen-treated animals, including animals treated with E2 with E2/raloxifene/aspirin, indicating an additive effect of the

alone, E2 plus raloxifene, and E2 plus aspirin, as well as com- combination therapy.

bined E2, raloxifene and aspirin, compared to vehicle controls The mechanism by which a target cell simultaneously in the OVX rabbits. However, maximal breast and uterine responds to a mixture of drugs is not entirely clear. Hypo-

stimulations were observed with E2 alone and E2 plus aspirin. thetically, the different molecules would compete for the avail- In contrast, raloxifene alone did not stimulate the uterus and able pool of receptors in the target cell, and simple receptor mammary glands, and when raloxifene was co-administered kinetics would dictate the dominant effector molecule. The

with E2, the stimulation of the breast and uterus was reduced final physiological response would be attributed to a blend of to vehicle control levels. all of the activities of these molecules. In the current study, Platelets contribute to thrombosis in several ways. They raloxifene was shown to abrogate the stimulatory effects of

provide the membrane surface for the generation of thrombin, E2 on the uterus and breast effectively, while aspirin reduced

express membrane receptors that affect platelet-platelet and the stimulatory effect of E2 on platelet aggregation, and both

platelet-vessel wall interactions. In low doses, aspirin has E2 and raloxifene demonstrated positive agonistic effects on an antiplatelet effect by inhibiting the production of throm- the lipid and MCP-1 profiles, and combined raloxifene, aspirin [30, 31] boxane. Platelets contain both ERα and ERβ , but the and E2 was the most efficacious treatment in reducing athero- effect of estrogen on the platelet aggregation has been contro- sclerotic lesions in the aorta. versial[32, 33]. In the current study, significant stimulation in The ideal postmenopausal estrogen therapy is expected to

platelet aggregation was observed with E2 alone and E2 plus reproduce the beneficial effects of estrogen without produc- raloxifene, but not with raloxifene alone in the OVX rabbits. ing the adverse effects; however, no ideal postmenopausal As expected, aspirin reduced the platelet aggregation signifi- estrogen therapy has been found to date. The present study cantly compared with vehicle control, and addition of aspirin demonstrated for the first time that the combination of ralox-

to E2 decreased the platelet aggregation to the vehicle control ifene, aspirin and E2 might be an attractive novel paradigm of levels. HRT. This finding may prompt additional research with the Due to the fact that raloxifene antagonized the stimulatory aim of improving quality of life for peri- and postmenopausal

effect of E2 on the breast and uterus and aspirin reduced the women.

stimulation of E2 in platelet aggregation, an obvious question that arose was whether the combinatory use of raloxifene and Acknowledgements

aspirin with E2 would unfavorably antagonize the positive This work was supported by Grant from the Natural Sci-

effects of E2, such as lowering levels of LDL-C and MCP-1 and ence Foundation of Shandong Province, Ji-nan, Shandong, inhibiting atherosclerotic lesions[1–5]. China (#Y2005C50), Grant-in-Aid for China-Japan Sasagawa It has been well-documented that a high level of LDL-C and Researchers, from the Ministry of Health, Beijing, China a low level of HDL-C are risk factors for atherosclerosis, and (#083). The authors are grateful to Chun-xi LIU and Zhi MCP-1, a potent chemoattractant for monocytes, has been sug- YANG for their excellent technical contributions. gested to be an especially important mediator of atherogenic signals[34, 35]. Raloxifene has been confirmed to have estrogen- Author contribution agonist effects on lipid profiles[14, 15] , and an study has Hong HE and Fa-lin YANG designed the research and wrote shown that raloxifene could downregulate the expression the paper; Ke-qing HU performed the research and analysed of MCP-1 in human coronary artery smooth muscle cells, the data; Hong HE, Fa-lin YANG, Xin WANG, Zi-mo LIU, although the degree of this inhibition was less than when Qin HU, and Ji-fu LI contributed to perform the research and induced by estrogen[36]. In the present study, ovariectomy analyse the data. induced a significant decrease in serum estradiol level and increased total cholesterol, LDL-C, and MCP-1 levels, as well References as decreased HDL-C levels. However, significant reductions 1 Ross R. Atherosclerosis-an inflammatory disease. N Eng J Med in total cholesterol, LDL-C, and MCP-1 levels and an increase 1999; 340: 115–26. 2 Blum A, Cannon Ro III. Effects of and selective oestrogen in HDL-C level were found with E2 treatment alone and ral- oxifene alone compared with the vehicle control, and further receptor modulators on serum lipoproteins and vascular function. Curr Opin Lipidol 1998; 9: 575–86. effects were observed with their co-administration, which sug- 3 Störk S, Baumann K, Clemens S, Angerer P. The effect of 17β-estra­ gested that raloxifene reinforced the positive effects of E . As 2 diol on MCP-1 serum levels in postmenopausal women. Cardiovasc well as its anticoagulant properties, aspirin has cardioprotec- Rev 2002; 53: 642–9. tive effects that include inhibiting the proliferation of vascular 4 Adams MR, Kaplan JR, Manuck SB, Koritnik DR, Parks JS, Wolfe MS, smooth muscle cells and improving endothelium-dependent et al. Inhibition of coronary artery atherosclerosis by 17β-estradiol [37] vascular relaxation . In the present study, reductions in the in ovariectomized monkeys: lack of an effect of added progesterone. sizes of atherosclerotic lesions were found by Oil Red O stain- Arteriosclerosis 1990; 10: 1051–7.

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